Proteomics, which entails global gene expression analysis, is considered a nonbiased discovery-driven (as opposed to hypothesis-driven) approach to the analysis of protein expression. The long-term objective of the proposed research is to determine the role of human bone marrow (BM) stromal cells in normal and leukemic hematopoiesis. This proposal aims to: (1) Carry forward a critical previous discovery that shows that human BM stromal cells represent a unique pluridifferentiated mesenchymal progenitor cell type (MPC), coexpressing multiple mesenchymal lineage markers. (2) Apply a recently established method for purifying MPCs from leukemic BM stromal cell cultures, free of macrophages and hematopoietic cells. (3) Investigate MPC cell-specific protein expression profiles and how these profiles change in hematopoietic malignancies. (4) Establish the proof of principle and practice of high-resolution proteomics with relevance to normal and leukemic human BM stromal cells. Specific experimental design includes performance of the following. R21 Phase: Set up large-format 2-D gel electrophoretic system (2-D PAGE) for reproducible separation of MPC proteins. Prepare 2-D PAGE protein maps for normal BM-derived MPCs (untreated and treated with representative cytokines) and for MPCs derived from patients with representative leukemic conditions (AML, CML, MM). Using mass spectrometry (MALDI-MS and/or Nano ESI MS/MS), identify about 200 differentially-expressed MPC proteins (i.e., those that increased or decreased in intensity as compared to 2-D PAGE protein maps of normal, unstimulated MPCs). Construct a human BM MPC-2DPAGE database on WWW and publish it under WORLD-2DPAGE with links to the currently existing 2-D PAGE databases. In parallel studies, identify about 200 differentially expressed MPC proteins by an independent method, isotope-coded affinity tag (ICAT) labeling in conjunction with LC/MS/MS. Based on theoretical pI and MW, construct a "virtual" 2-D map of ICAT-identified MPC proteins for integration into 2-D PAGE database. R33 Phase: Add high-throughput robotics to the high-resolution 2-D PAGE proteomics established under R21 phase. Identify on a relatively large-scale (about 1,800) the MPC proteins that are differentially expressed following stimulation with different cytokines and in leukemias (AML, CML, MM). Facilitate understanding of the pathogenetic mechanisms by identifying the phosphoproteins potentially involved in cell signaling pathways. In parallel studies, identity about 1,800 MPC proteins by ICAT method. We plan to identify a total of about 2,000 functionally relevant BM stromal cell proteins by each method. Update the WWW database by including the proteins with pathologically altered expression. The database will be a valuable resource for researchers investigating the basic biology of hematopoiesis and leukemogenesis and for clinical hematologists/oncologists and pathologists alike.